Cd3e fitc

Cells were stained and analyzed on either a FACSCalibur II or LSR II (Becton Dickinson, San Jose, CA). The following anti-mouse antibodies were used for flow cytometry analysis: B220-PE, CD3e-FITC, CD11b-APC, Gr-1-APC-Cy7, CD48-Pacific Blue, CD150-PE-Cy7, CD34, c-Kit-APC, Sca-1-PE, FcγR-PE, CD45.1-Brilliant Violet 570, and CD45.2-Alexa Fluor 700 (all from eBiosciences, San Diego, CA). Cell sorting was performed using a FACS Aria (Becton Dickinson, San Jose, CA). Antibodies for Annexin V and Ki-67 assays were used as per manufacturer’s protocol (Becton Dickinson, San Jose, CA). Gating strategy for HSPC fractions are as follows: (1) LSK: Lineage^NegCkit+Sca1+; (2) CMP: Lineage^NegCkit+Sca1^Neg CD34+FcγRIII^low; (3) GMP: Lineage^NegCkit+Sca1^Neg CD34+FcγRIII^high; (4) MEP: Lineage^NegCkit+Sca1^Neg CD34^NegFcγRIII^Neg; (5) CLP: Lineage^Neg IL7Rα^highCkit+Sca1^low; ProB Frac A: B220^lowCD43^HighBP1^NegHSA^Neg; ProB Frac B: B220^lowCD43^HighBP1^NegHSA^Pos; ProB Frac C: B220^lowCD43^HighBP1^HighHSA^Pos; ProB Frac D: B220^HighCD43^NegIgM^Neg; ProB Frac E: B220^HighCD43^NegIgM^Mid; ProB Frac F: B220^High++CD43^NegIgM^High/Mid.

Splenocytes of three mice vaccinated by KP-LC and 3 mice vaccinated by PBS were stained with CD3e (FITC) (eBioscience, 11-0031-86), CD4 (APC) (eBioscience, 17-0041-82), CD8a (PE) (eBioscience, 12-0081-85), CD44 (eFluor 450) (eBioscience, 48-0441-80), CD62L (PerCP-Cyanine5.5) (eBioscience, 4300748) for 30 min at 4°C. After washing, stained cells were analyzed on an ACEA NovoCyte flow cytometer.

Orthotopic Met-1 or AT3 tumours were isolated, dissociated into single cell suspension, and incubated for 15 min with anti-mouse CD16/CD32 to reduce non-specific staining. Infiltration of immune cells into tumours was analysed by staining with the following anti-mouse antibodies: CD45-PE/Cy7 (eBioscience, 25-0451-82, diluted 1:200), CD45-BV570 (Biolegend, 103136, diluted 1:100), CD3e-FITC (eBioscience, 14-0031-82, diluted 1:100), CD4-APC/Cy7 (Biolegend, 100414, diluted 1:100), anti-CD8a-APC (eBioscience, 17-0081-82, diluted 1:100), CD11b-PerCP/Cy5.5 (eBioscience; 45-0112-82, diluted 1:100), F4/80-APC (eBioscience, 17-4801-82, diluted 1:50), Ly-6G/Ly-6C (Gr-1)-APC/Cy7 (Biolegend, 108424, diluted 1:100), Ly-6G-APC (Biolegend, 127614, diluted 1:200), Ly-6C-FITC (Biolegend, 128006, diluted 1:200), Ly-6C-BV605 (Biolegend, 128036, diluted 1:200). DAPI was used to exclude dead cells. Acquisition was performed with Beckman-Coulter Gallios flow cytometer and data analysis was done with FlowJo software (version X.0.7).

Ascites samples from the orthotopic ID8 OC model were spun at 300 g for 10 min to separate cellular and acellular fractions. Ascites cell pellets were incubated for 5 min with 5 mL of red cells lysis buffer (Versalyse lysing solution; #A09777, Beckman Coulter, Villepinte, France) and then washed with PBS. Pelleted cells were suspended in freezing medium (90% FBS/10% DMSO) and kept in liquid nitrogen for subsequent analysis. After thawing, cells were re-suspended in RPMI 1640 medium containing 10% FBS and 0.5% C₃H₃NaO₃. Viable cells were then gated based on forward and side scatter profiles, and equal numbers of viable ascites cells were phenotyped, using the following fluorescent-labeled antibodies: CD3e-FITC (#11-0031-82, Thermo Fisher Scientific), CD45R-APC (#17-0452-82, Thermo Fisher Scientific), CD4-PE (#12-0041-82, Thermo Fisher Scientific) and CD8a-PerCP-Cyanine5.5 (#45-0081-82, Thermo Fisher Scientific) for 20 min, at room temperature. After washing with DPBS (#D8537, Sigma-Aldrich), stained samples were run on a BD Accuri™ C6 cytometer (Beckman Coulter) followed by gating, data analysis and visualization in FlowJo^TM (FlowJo LLC, Ashland, OR, USA) software.